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Abstract Primer pairs were designed to amplify the flanking region of known gene by 2-3 round of PCR，3 nested primers ( p14, p15, p16) and a group of 9 restriction site primers in total were designed. Each restriction site primer consist of a random primer p17 plus a restriction site sequence at 3’ end, 3 nested primers were designed based on the sequence of known gene. First round of PCR was performed using outer nested primer p14 and 9 restriction site primers，its 5 initial cycles of amplification at low annealing temperature allows the incorporation of 5’ sequence of restriction site primer into the PCR products，then the annealing temperature of subsequent cycles of PCR can be increased. The later cycles were performed at a higher annealing temperature so that the entire restriction site primer could stably anneal only to PCR products from the initial cycles. In order to increase specificity and yield of the amplification, the primers p15 and p17 were used to conduct second round of PCR, primers p16 and p17 were used to conduct third round of PCR, the PCR products were purified and sequenced. In order to obtain correct sequence, new primer f3x was designed using sequenced DNA, then isolated DNA as model, primers f3x and p14 (or p15) were used to perform PCR and products sequencing. Using this method, the 528bp promoter and 142bp 5’UTR sequence of FPPS gene was obtained. The sequence has been registered in the GenBank (accession no. FJ2263960). It was found by Blastn tool that this is the first report of the FPPS gene promoter sequence. These results indicted that this is a suitable method for amplifying the flanking region of known gene.
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Received: 01 December 2008
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